1. Field of the Invention
The present invention relates to a technology for detecting a target substance by using evanescent light. Specifically, the present invention relates to, for example, a technology for fluorescence detection of a large number of DNAs, proteins, or the like by using evanescent light.
2. Description of the Related Art
As a means for detecting a target substance, such as DNA or protein, there is a widely-used method including: attaching a fluorescent label to a target substance; irradiating the labeled target substance with predetermined excitation light, such as a laser; and detecting the fluorescence emitted from the labeled target substance. The methods for detecting fluorescence with high contrast include a detection method using, as excitation light, evanescent light generated by total internal reflection. In this method, a labeled substance on the front surface of a fused silica is detected using, as the excitation light, evanescent light which is generated, from a laser beam incident from the rear surface of the fused silica, at an interface between the front surface of the fused silica and a solution layer. Generally, the intensity of evanescent light decays exponentially as the evanescent light travels further away from a refractive index interface plane, and decays to 1/e at a distance of around 150 nm from the refractive index interface plane. Accordingly, in the fluorescence detection using the evanescent light as the excitation light, an effect of the fluorescence emitted from a material other than the target substance to be detected, can be reduced significantly, and as a result, the fluorescence detection can be carried out with high contrast.
In P. N. A. S. 2003, Vol. 100, pp. 3960-3964 (Non-Patent Document 1), DNA elongation is detected using the above-described method. Specifically, first, a primer labeled with a Cy3 molecule is supplied onto a fused silica where a single DNA molecule is fixed by the protein binding of biotin-avidin. The positional information on the target DNA is recognized from the fluorescence intensity obtained by hybridization of the supplied primer and the target DNA molecule. Subsequently, DNA elongation of the primer molecule is performed, and then the DNA elongation is specifically detected from the positional information and the fluorescence generated from a dNTP molecule that is introduced in the DNA elongation.
In Anal. Chem. 2003, 75, pp. 6629-6633 (Non-Patent Document 2), more sensitive fluorescence-detection has been attempted using a plasmon resonance that is caused by introducing a silver thin film onto a fused silica. The plasmon resonance is a phenomenon of the resonance that is caused by the coupling of free electrons localized in a metal with an oscillating electric field of incident light. An enhanced electric field generated by the resonance causes enhanced excitation light and fluorescence, and as a result, highly sensitive fluorescence-detection can be expected. As such a metal that provides a strong resonance in a visible range, gold, silver, and the like are known.
On the other hand, in U.S. Pat. No. 6,917,726 (Patent Document 1), the fluorescence of a target substance is detected using a near-field light coming from an opening portion formed in an aluminum thin film expected to produce a light shielding effect, the opening portion sufficiently smaller than the wavelength of excitation light.